The major objective is essentially unchanged from the original submission, viz. to develop a live oral vaccine for the prevention of cholera using recombinant DNA techniques. We have used these techniques to delete a single virulence factor, cholera toxin, from fully virulent V. cholerae which had been extensively characterized in volunteers. We derived isogenic, cholera toxin negative vaccine strains which elicited an excellent immune response in vaccinees. This immunization provided strong protection against challenge with the fully toxinogenic parent at a vaccine efficacy equal to that generated by clinical cholera infection. Unexpectedly, vaccinees experienced mild to moderate diarrhea on initial immunization. We propose to further attenuate these vaccine strains by the selective cloning and deletion of other possible virulence factors. This genetic approach to vaccine development offers a unique opportunity to manipulate a single virulence determinant and retain the antigens which elicit protective immunity. The power of recombinant DNA techniques, however, is limited by the lack of knowledge as to the critical combination of antigens required to elicit protective immunity to cholera. V. cholerae produces a number of substances which some evidence suggests may function as secondary virulence factors. We propose to construct isogenic vaccine candidates lacking these factors and characterize them to evaluate their potential as vaccines. In addition to determining the usefulness of these strains as immunizing agents, this investigation should yield valuable information regarding V. cholerae pathogenesis. We have already isolated clones containing genes encoding many of these factors from gene libraries of V. cholerae constructed dure the first two years of this research. Specifically, we propose to sequentially delete the following virulence factors from both classical and El Tor vaccine strains: 1) the El Tor hemolysin, 2) the vibrio Shiga-like toxin, 3) the recently isolated cytonic hemolysin, 4) the fucose-sensitive HA, and 5) the protease/HA. In addition, we propose to derive a) nonflagellated and b) nonmotile, flagellated mutants of V. cholerae. All strains generated above will be examined using animal models as a preliminary assessment of their potential as vaccine candidates. In addition, antigenic differences between attenuated strains and virulent parents will be characterized by immunoblot analysis.